During the current grant period we have focused on the development of conformationally constrained, receptor selective opioid ligands for the elucidation of the key structural elements required for receptor binding and their relative orientation in space (the ligand pharmacophore). This has led to pharmacophore models for two closely related tetrapeptide series, one selective for the d opioid receptor, one selective for the m receptor. To complement this ligand directed focus, we have also developed structural models of opioid receptors and other G protein-coupled receptors (GPCR5). Combining these two areas has led to the development of ligand-receptor interaction models for these two closely related peptide series at the m and d receptors and allows us to propose specific features of the ligands and their receptors that underlie the ligands' selectivities. We propose to extend these observations and test and refine our hypotheses for ligand-receptor interaction models for the mu and delta opioid receptors. This will be done through evaluation of carefully design receptor mutations with known or newly designed complementary ligands and will test specific predictions of our interaction models. Extension of the models to include kappa and ORL1 receptors is also planned. We will also employ our ligand receptor interaction models for the design of new ligands based on our previously developed scaffolds, employing each scaffold for the design of ligands for each opiold receptor. This would provide convincing support of both our receptor models and our ligand-receptor interaction models. The scaffolds to be elaborated are the cyclic tetrapeptide framework used to develop structurally similar ligands selective for mu and delta receptors and a peptidomimetic scaffold that was itself designed based on our pharmacophore models. This peptidomimetic scaffold has already yielded very potent, modestly selective ligands for the mu receptor. In addition to these opioid ligand - opioid receptor studies, we will extend our exciting preliminary studies ultimately aimed at augmenting agonist action at GPCRs by inhibition of RGS (regulators of G protein signaling) protein acceleration of Ga GTPase activity, which serves to terminate the agonist induced transduction.